Comprehensive, High-Spatiotemporal-Resolution Intravital Two-Photon Imaging of the Mouse Conventional Outflow Pathway: Green Intravital Imaging

Purpose: To develop an intravital imaging method that comprehensively shows the mouse conventional outflow pathway at high spatiotemporal resolution and to investigate the fine structures and dynamics within this pathway. Methods: Intravital two-photon imaging was performed using Green, Prox1-reporter, and mito-Dendra2 mice. Schlemm's canal (SC) in human patients with glaucoma was histologically evaluated. Results: The intravital two-photon imaging of Green mice ubiquitously expressing enhanced green fluorescent protein (EGFP) ("Green intravital imaging") demonstrated the perilimbal vein (PLV), collector channels (CCs), SC, and trabecular meshwork with and without a coverslip. A wide, tubular structure with septa was identified as SC in Prox1-reporter mice. Structures reminiscent of giant vacuoles and EGFP-positive vesicle flow were observed within SC. Based on the vesicle size, mitochondria in the vesicles, and platelet-specific intravital imaging findings, these EGFP-positive vesicles were identified as platelets. Moreover, platelet flow from the PLV into SC and from SC back to the PLV via CCs, as well as fast aqueous humor flow converging toward CCs, was observed. The quantitative analysis results suggested differences in aqueous flow velocity and flow patterns within SC. Platelets were also observed in SC of patients with glaucoma. Conclusions: Green intravital imaging comprehensively showed the proximal and distal conventional outflow pathway in mice at submicrometer spatial and 64.9-ms/frame temporal resolution, allowing observation of structures reminiscent of giant vacuoles. A subset of platelets transiently entered and exited SC via CCs. Within SC, aqueous humor exhibited fast flow converging toward the CCs, with differences in flow velocity and patterns. CCs may be involved in the heterogeneous dynamics of the aqueous humor.